Pepper plant

ABSTRACT

The present invention generally relates to seedless pepper (SLP); and more specifically, it pertains to male sterile pepper plants having unique characteristics including the capability of growing edible seedless fruits, wherein the “seedless” trait is controlled by a genetic determinant independent of exogenous factors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/663,606, filed May 17, 2010 (now U.S. Pat. No. 8,492,619), whichclaims priority under 35 U.S.C. §371 from International Application No.PCT/EP2008/057486, filed Jun. 13, 2008, which claims the benefit ofIsraeli Application No. 183902, filed Jun. 13, 2007, the disclosures ofwhich applications are incorporated by reference herein in theirentirety.

The present invention generally relates to seedless pepper (SLP); andmore specifically, it pertains to male sterile seedless pepper havingunique characteristics as set forth below.

Fruits are the result of the development of the ovary. In normal fruitdevelopment, the initiation of the fruit set depends on the successfulcompletion of the pollination and fertilization process. Initiation offruit development is generally repressed until fertilization occurs.

In general, the entire development of the fruit follows pollination andfertilization, and coincides with the maturation of the seeds. A fruitprovides a suitable environment for the development of seeds.

External or internal factors may occasionally interfere with the normalprocess of fruit development, which then may become independent ofpollination and fertilization such as, in parthenocarpic fruit setting.In parthenocarpic plants, fruit development and initiation is uncoupledfrom fertilization. The parthenocarpic characteristic can lead to thedevelopment of fruits with no or reduced seeds without pollination andfertilization. Parthenocarpy therefore refers to the development of theovary into seedless fruit independent of the pollination and/orfertilization process.

The parthenocarpic characteristic is well known in many plant speciesand may occur naturally or can be induced artificially with a variety ofexternal stimuli such as, for example, application of differenthormones. In most of the species the source of the parthenocarpicphenomenon, which results in the production of fruits with no or reducedseeds, may have a genetic and/or epigenetic basis. Parthenocarpy inplants may be seen as a mutation that reduces the plant's chances ofsurvival and multiplication. One would logically assume that plants“want” to save their energy only for fruits that raise their chances ofmultiplication, and that, therefore, evolution would seek to eliminateplants producing seedless fruits or initiated fruits may drop in case nofertilization takes place. This is perhaps the reason why theparthenocarpic phenomenon is so rare. In crop cultivation, it is usedfor essentially two purposes: A): to produce seedless fruits where seedsare viewed as undesirable (watermelon, cucumber) and B): to increaseyield when fertilization conditions are adverse (tomato, eggplant).

Parthenocarpy is frequently accompanied by sterility, as in watermelon,but the characteristic is also found as an independent trait such as,for example, in cucumber and tomato.

Parthenocarpy can occur naturally or it can be artificially induced.Natural parthenocarpy may have a genetic basis and therefore becontrolled by genetic determinants. The “seedless” trait can beobligatory or optional, that is it may be expressed all times ordepending on the environmental conditions.

An artificially induced parthenocarpy may be the result of treatment ofthe plant and/or flower and/or ovary with exogenous agents such as deadpollen extract, or of applying growth regulating substances, eithernatural or synthetic. In fact, in protected cultivation such as, forexample, in the greenhouse, exogenous applications of growth regulatorsare frequently used in order to induce the formation of seedless fruitswith the aim of improving yield and fruit quality, but also in openfield cultivation to prevent production losses under adverse conditions.However, these measures are cost and labor intensive and often also leadto only partial parthenocarpy. Furthermore, chemical treatment may alsoresult in an increase of chemical residues on the fruits and in the soiland also malformations of the fruits are often to be observed.

Seedless sweet pepper cultivars based on natural parthenocarpy are notyet available; therefore, chemical manipulation was used to artificiallyinduce parthenocarpy. Auxins, gibberellins and cytokinins (Sjut andBangerth, 1982/83; Kim et al., 1992), as well as auxin transportinhibitors (Beyer and Quebe-deaux, 1974; Kim et al., 1992), have allbeen successfully applied to several fruit vegetable crops.

Fruit quality and yield are commonly dependent on the amount of seedspresent in the fruit. High numbers of seeds usually lead to an increasein fruit size and quality. Pepper fruit yield and quality isparticularly well known as being highly dependent on the amount ofseeds. Moreover, pepper is usually very sensitive to setting. Once theplant starts this process it turns the majority of its energy tosetting. As a result of this competition for limited energy andassimilate resources, subsequent set and growth of fruits is inhibitedas well as growth of leaves, branches and roots. An increasing number ofseeds reduce plant growth further.

This sensitivity of pepper plants to setting is one of the reasons forthe “flushing” phenomenon, which leads to cyclic fluctuations of fruitset and thus in pepper fruit yield with weeks of high yield alternatingwith weeks of low yield. This irregular harvest pattern is an importantproblem for pepper growers because it makes it difficult for the growersto meet the market demand during growing seasons and further leads tofluctuations in labor demand in the greenhouses.

In other species, tomato for example, the sensitivity to setting is muchlower. Tomatoes can produce relatively good yield and quality fruitseven when they have few seeds, or continue to grow even when the fruitshave a large amount of seeds. Artificial tools can ensure fruit settingin some species where fertility conditions are adverse. In tomato,spraying of a special hormone (auxin) can induce fruit setting, evenwithout seeds.

Heuvelink and Körner (2001) tested the working hypothesis that growingparthenocarpic fruit on pepper plants may at least reduce the flushingproblem in pepper growth and further yield high quality pepper fruits.

Heuvelink and Körner (2001) induced parthenocarpic fruit growth inpepper by preventing self-pollination and applying auxin to the stigma.They could confirm that parthenocarpic fruit growth resulted in a moreregular fruit set and yield and thus has the potential to reduce theproblem of flushing.

Induction of parthenocarpic growth by external application of planthormones or other inducing agents is, however, labour and cost intensiveand may raise environmental concerns due to increased amounts ofchemical residues on or in the fruits or the soil.

This approach is therefore not practical for commercial pepperproduction. There was therefore a need for genetic solutions that avoidcyclic fluctuations in pepper yield, but would instead guarantee acontinuous supply with high quality pepper fruits.

The present invention now offers such a solution which is suitable forcommercial use by providing a parthenocarpic pepper plant which growsseedless fruits, wherein setting of seedless fruits is independent ofthe pollination and fertilization process and/or of treatment withexogenous agents, but controlled by a genetic determinant that is stableand thus can be stably inherited to progeny plants and introgressed intocommercial pepper varieties.

This invention also offers the advantage of improved options for insectand disease control. Viral diseases of pepper are generally transmittedby aphids. These insects can be controlled with the use of mechanicalmeans such as tissue nets. These insect control measures have, however,the disadvantage that also beneficial insects are prevented fromaccessing the pepper plants and therefore no insect-mediated pollinationcan occur. The present invention now permits protecting pepper plantsfrom aphid infestations and thus from aphid-transmitted virus infectionswithout interfering with normal fruit setting and fruit harvest andwithout the need for chemical pesticides, thus reducing the costs ofinsect and disease control.

In particular, the present invention provides a pepper plant, whichgrows seedless fruits, wherein the “seedless” trait is controlled by agenetic determinant independent of the pollination and fertilizationprocess and also independent of treatment with parthenocarpy-inducingplant hormones including auxins, gibberellins and cytokines, auxintransport inhibitors, or others and/or other parthenocarpy-inducingexogenous factors and/or exogenously administered parthenocarpy-inducingagents such as growth regulating substances, either natural orsynthetic, or plant extracts such as, for example, dead pollen extract.

In one embodiment of the invention, a pepper plant is provided, whichgrows seedless fruits, wherein the “seedless” trait is controlled by agenetic determinant and independent of exogenous treatment withparthenocarpy-inducing plant hormones.

In one embodiment, said pepper plant according to the invention growsseedless fruits independent of treatment with plant hormones selectedfrom the group consisting of auxins, gibberellins and cytokines, auxintransport inhibitors, and others.

In one embodiment of the invention, a pepper plant is provided, whichgrows seedless fruits, wherein the “seedless” trait is independent ofthe pollination and fertilization process.

In one embodiment, said pepper plant according to the invention growsseedless fruits, which are at least 95%, particularly at least 98%,particularly at least 99% seedless.

In one embodiment, said pepper plant according to the invention growsseedless fruits, which are 100% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 40%, particularly at least50%, particularly at least 60%, particularly at least 70%, particularlyat least 80%, particularly at least 90%, but especially 100% of thefruits grown on said plant are seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 40%, particularly at least50%, particularly at least 60%, particularly at least 70%, particularlyat least 80%, particularly at least 90%, but especially 100% of thefruits grown on said plant are seedless fruits, which are at least 95%,particularly at least 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 40% of the fruits grown onsaid plant are seedless fruits, which are at least 95%, particularly atleast 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 60% of the fruits grown onsaid plant are seedless fruits, which are at least 95%, particularly atleast 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 70% of the fruits grown onsaid plant are seedless fruits, which are at least 95%, particularly atleast 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 80% of the fruits grown onsaid plant are seedless fruits, which are at least 95%, particularly atleast 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 90% of the fruits grown onsaid plant are seedless fruits, which are at least 95%, particularly atleast 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 95% of the fruits grown onsaid plant are seedless fruits, which are at least 95%, particularly atleast 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein at least 99% of the fruits grown onsaid plant are seedless fruits, which are at least 95%, particularly atleast 98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 100% of the fruits grown on saidplant are seedless fruits, which are at least 95%, particularly at least98%, particularly at least 99% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 40% of the fruits grown on said plantare seedless fruits, which are 100% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 60% of the fruits grown on said plantare seedless fruits, which are 100% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 80% of the fruits grown on said plantare seedless fruits, which are 100% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 90% of the fruits grown on said plantare seedless fruits, which are 100% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 95% of the fruits grown on said plantare seedless fruits, which are 100% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 98% of the fruits grown on said plantare seedless fruits, which are 100% seedless.

In one embodiment, a plant according to the invention and as describedherein before is provided, wherein 100% of the fruits grown on saidplant are seedless fruits, which are 100% seedless.

In particular, the present invention provides a pepper plant accordingto the invention and as described herein, which grows seedless fruitswhich are edible and of high quality.

In one embodiment, the present invention provides a pepper plant growingseedless fruits which are edible and of high quality and suitable ofbeing used as fresh produce, as fresh cut produce, or for processingsuch as, for example, canning.

In one embodiment, said pepper plant according to the invention iscapable of setting seedless fruits throughout the plant. In particular,fruit setting starts at the first nodes of a branch and progresses alongthe length of the entire branch. In one embodiment, said plant growsparticularly two fruits per node in about 20%, particularly in about40%, particularly in about 60% of the nodes.

In one embodiment, said pepper plant according to the invention showsfruit setting at all seasons that is also under unfavourable conditionswithin the context of the climatic conditions of Israel or comparableclimes.

In one embodiment, said pepper plant according to the invention growsnormal looking fruits, which comply with commercial quality standardssuch as defined, for example, in the United States Standards for Gradesof Peppers (USDA, Agricultural Marketing Service, Fruit and VegetablePrograms, Fresh Products Branch).

In still another aspect of the invention, the plant according to theinvention and as described herein before carries fruit, which, atmaturity, weigh over 2 grams or are longer than 1 cm and have a diameterof over 0.5 cm, when said plant is grown under growing conditionsgenerally used by growers in regular cropping practice, in open field orin protected cultivation.

The pepper plant according to the invention and as described hereinbefore may grow a sweet pepper including a dolce-type pepper, a bellpepper, a big rectangular pepper, a conical pepper, a long conicalpepper or a blocky-type pepper. The fruit of said plant at maturity maybe an evergreen, a yellow, orange, ivory, brown, purple, or red fruit.

The plant according to the invention may be a hot pepper plant, e.g. amildly pungent pepper used for the fresh market and for processingincluding the long, heart-shaped, thin-fleshed Ancho-type and the long,blunt-ended, thin-fleshed Tuscan-type pepper, the slightly more pungentChili pepper fruit with medium flesh thickness, and a pungent pepperused in both the fresh market and for processing including the long,cylindrical-thick fleshed Jalapeno, the small, slender, tapering Serranoand the irregularly shaped, thin-fleshed Cayenne pepper.

The plant according to the invention and as described herein before maybe an inbred, a dihaploid or a hybrid and/or a male sterile.

In one embodiment, said pepper plant according to the invention and asdescribed herein is male sterile.

In one embodiment, a pepper plant is provided, particularly a seedlesspepper plant according to the invention and as described herein, whichgrows pepper fruits which are red pigmented in the mature and dark greenpigmented in the premature (unripe) stage.

In one embodiment of the invention, said pepper fruits at maturity havevery sweet taste with a Brix of between about 7° to about 14°,particularly of between about 7.5° to about 12°, particularly of betweenabout 8° to about 11°.

In one embodiment of the invention, said pepper fruits have a conic-likeshape, i.e., between bell and classic conic and a size of between about2 to 4 cm by about 3 to 4 cm in diameter.

In one embodiment, a pepper plant is provided, particularly a seedlesspepper plant according to the invention and as described herein, whichgrows a pepper fruit which is red pigmented in the mature and dark greenpigmented in the premature (unripe) stage; have very sweet taste with aBrix of between about 7° to about 14°, particularly of between about7.5° to about 12°, particularly of between about 8° to about 11°; have aconic-like shape, i.e., between bell and classic conic and a size ofbetween about 2 to 4 cm by about 3 to 4 cm in diameter.

In one embodiment, the pepper plant according to the invention and asdescribed herein before contains a “seedless” trait, which is obtainablefrom a hybrid pepper plant selected from the group of hybrids consistingof Capsicum annuum AR07-F1-56-b; Capsicum annuum AR07-F1-87-b; Capsicumannuum AR07-F1-166-b; Capsicum annuum AR07-F1-171-X; and Capsicum annuumAR07-F1-172-X. Seeds for growing such hybrid plants have been depositedwith NCIMB, Aberdeen AB21 9YA, Scotland, UK on May 26, 2008 underaccession number NCIMB 41558, NCIMB 41559, NCIMB 41560, NCIMB 41561 andNCIMB 41562, respectively.

In one embodiment of the invention, the “seedless” trait or a plantcomprising said trait is obtainable from any of the hybrid lines grownfrom the deposited seeds by growing the F2 progeny of said hybrid. Inparticular, the “seedless” trait or a plant comprising said trait isobtainable from any of the deposited hybrid lines by i) germinating seedof said lines and growing a mature, fertile plant therefrom; ii)inducing self-pollination of said plant grown under (i), growing fruitsand harvesting the fertile seeds therefrom, and iii) growing plants fromthe seeds harvested under ii) and selecting plants which grow seedlessfruits.

In one embodiment, the invention relates to plant material obtainablefrom a plant according to the invention and as described herein beforeincluding, but without being limited thereto, leaves, stems, roots,flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds,cuttings, cell or tissue cultures, or any other part or product of theplant which still exhibits the seedless phenotype according to theinvention, particularly when grown into a plant.

The invention further relates to plant parts obtainable from a plantaccording to the invention and as described herein before including, butwithout being limited thereto, plant seed, plant organs such as, forexample, a root, stem, leaf, flower bud, or embryo, etc, ovules, pollenmicrospores, plant cells, plant tissue, plant cells cultures such as,for example, protoplasts, cell culture cells, cells in plant tissues,pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos atvarious stages of development, etc; which still exhibits the seedlessphenotype according to the invention, particularly when grown into aplant.

The invention further relates to an agronomic method of producingseedless pepper fruits comprising the steps of

-   -   i) providing a pepper plant according to the invention and as        characterized herein before;    -   ii) multiplicating/propagating said pepper plant    -   iii) optionally preventing pollination of said pepper plant by        using, e.g., male fertile pepper plants, functional male sterile        pepper plants, or emasculation;    -   iv) allowing the plant to grow seedless pepper fruits; and    -   v) harvesting said pepper fruits.

In one embodiment of the invention, the multiplication or propagation ofthe pepper plant is done either through seeds or by vegetativepropagation.

The invention further relates to a method of producing a seedless pepperplant comprising the steps of

-   -   i) providing seeds of an F1 hybrid made by crossing of a        seedless pepper plant as a female line and a male-fertile        (seed-bearing) pepper plant as a male line;    -   ii) germinating said seed and growing a mature, fertile plant        therefrom;    -   iii) inducing self-pollination of said plant grown under (ii),        growing fruits and harvesting the fertile seeds therefrom, and    -   iv) growing plants from the seeds harvested under iii) and        selecting plants which grow seedless fruits.

In one embodiment, the hybrid seed used in said method according to theinvention is the hybrid seed, which is obtainable from a hybrid pepperplant selected from the group of hybrids consisting of Capsicum annuumAR07-F1-56-b; Capsicum annuum AR07-F1-87-b; Capsicum annuumAR07-F1-166-b; Capsicum annuum AR07-F1-171-X; and Capsicum annuumAR07-F1-172-X, grown from seeds deposited with NCIMB, Aberdeen AB21 9YA,Scotland, UK on May 26, 2008 under accession number NCIMB 41558, NCIMB41559, NCI MB 41560, NCI MB 41561 and NCI MB 41562, respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: SLP plant in the net house, Netiv Haasara village Israel. The“one bite” type variety “AR06-F3-255-1”. This variety was used as SLPsource in the deposited hybrids.

FIG. 2: Fruits from Yellow blocky SLP variety “SD07-3-5”, as resultingfrom the hybrid made between “AR06-F3-255-1” and a F2 plant of a yellowblocky pepper variety.

FIG. 3: Fruits from Red conic SLP variety “SD07-2-77”, as resulting fromthe hybrid made between “AR06-F3-255-1” and a F2 plant of a red Kapyatype pepper variety.

DEFINITIONS

The technical terms and expressions used within the scope of thisapplication are generally to be given the meaning commonly applied tothem in the pertinent art of plant breeding and cultivation if nototherwise indicated herein below.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a plant”includes one or more plants, and reference to “a cell” includes mixturesof cells, tissues, and the like.

“Parthenocarpy”, as used herein, refers to the production of fruitswithout fertilization and it permits seedless fruits to be obtained.Parthenocarpy is favored by certain environmental conditions such ashigh or low daytime or nocturnal temperatures, a low level of light andhigh humidity. Parthenocarpy can occur naturally or it can beartificially induced. Natural parthenocarpy is produced by (epi)geneticcauses and can be obligatory or optional, in other words, depending onthe environmental conditions.

“Seedless pepper fruit”, as used herein, refers to a pepper fruitobtained independently of the pollination and/or fertilization process.and/or independent of treatment with parthenocarpy-inducing planthormones and/or other parthenocarpy-inducing exogenous factors and/orexogenously administered parthenocarpy-inducing agents.

A seedless pepper fruit which is “XX %” seedless, refers to a fruitwherein ony “100−XX %” of the ovaries present in said fruit develop intoseeds. For example, a seedless pepper fruit which is “95%” seedless,refers to a fruit wherein ony “100−95%” of the ovaries present in saidfruit, that is 5% of the ovaries, develop into seeds.

A “seedless pepper plant” as used herein, refers to a pepper plantgrowing a seedless pepper fruit as defined herein, including a pepperplant wherein only “100−XX %” of the fruits present on said pepper plantare seedless, particularly 95% and up to 100% seedless.

As used herein, “edible fruit” and “fresh edible fruit” refer to fruitpicked from the plant that are suitable for human consumption.

As used herein, “exogenous factors or agents” refer to factors or agentsthat upon exogenous application to the plant, particularly to thereproductive parts of the plant such as, for example, the stigma, arecapable of inducing an artificial parthenocarpy in the treated plant.Such “exogenous factors or agents” may be, for example, dead pollenextract, growth regulating substances, either natural or synthetic suchas plant hormones including auxins, gibberellins and cytokines, auxintransport inhibitors, or others.

As used herein, the term “trait” refers to characteristic or phenotype,e.g., mature fruit color or a disease resistance such as TSWVresistance. A trait may be inherited in a dominant or recessive manner,or in a partial or incomplete-dominant manner. A trait may be monogenic(i.e. determined by a single locus) or polygenic (i.e. determined bymore than one locus) or may also result from the mutual interactionamong genes or interaction of one or more genes with the environment. Adominant trait results in a complete phenotypic manifestation atheterozygous or homozygous state; a recessive trait manifests itselfonly when present at homozygous state.

The term “pericarp” as is known in the art refers to the wall of amatured ovary. Specifically, pepper fruit pericarp refers to the fruitwall, which is the colored, edible part of the pepper fruit. As usedherein, the term “thick pericarp” refers to a pericarp width of at least5 mm, preferably at least 8 mm. As used herein the term “selfing” refersto a controlled self-pollination of a plant, i.e. contacting pollen andovule produced by the same plant. The term “crossing” refers tocontrolled cross-pollination, i.e. contacting pollen and ovule eachproduced by a different plant.

The term “plant vigor” is used herein in its broadest sense, referringto the plant general strength.

As used herein, a “fruit crop” refers to the crop of a single plant, orpreferably, to the fruit crop obtained from pepper plants grown on acommercial scale.

The term “commercial pepper line or variety” as is used herein refers toa pepper plant which is available commercially such as, for example,pepper variety having sweet, edible fruit with a thick pericarp.Typically, the commercial pepper variety may have fruit with a blocky,Californian-type fruit shape. Examples of commercial pepper varietiesinclude, without however being limited to, for example sweet peppercommercial cultivars of the “bell” type such as Cannon (Zeraim Gedera);Vergasa (Syngenta Seeds); Bardenas (Syngenta Seeds), Roxy (SyngentaSeeds), Yolo Wonder (Syngenta Seeds), etc.

“Low temperature” with regard to pepper plant growth according to thepresent invention refers to temperature in the range of 10°-12° C. andbelow depending on the variety used.

As used herein, the term “allele(s)” means any of one or morealternative forms or variant forms of various genetic units identical orassociated with different forms of a gene or of any kind of identifiablegenetic element, all of which alleles relate to at least one trait orcharacteristic. In a diploid cell, the two alleles of a given geneoccupy corresponding loci on a pair of homologous chromosomes and are,therefore, alternative in inheritance.

Such alternative or variant forms may be the result of single nucleotidepolymorphisms, insertions, inversions, translocations or deletions, orthe consequence of gene regulation caused by, for example, by chemicalor structural modification, transcription regulation orpost-translational modification/regulation.

In some instances it may be more accurate to refer to “haplotype” (i.e.a haplotype is a combination of alleles at several or multiple linkedloci (on the same chromosome) that are transmitted together) instead of“allele”, however, in those instances, the term “allele” should beunderstood to comprise the term “haplotype”. Alleles are consideredidentical when they express a similar phenotype, but in some cases itcan happen that different alleles also express a similar phenotype.Differences in sequence are possible but of less importance as long asthey do not influence phenotype.

An allele associated with a quantitative trait may comprise alternativeor variant forms of various genetic units including those that areidentical or associated with a single gene or multiple genes or theirproducts or even a gene disrupting or controlled by a genetic factorcontributing to the phenotype represented by said QTL.

A “genetic determinant” is defined herein as a nucleotide sequence,preferably a DNA sequence that may comprise sequences with variousgenomic functions such as genes and regulatory elements regions. Geneticdeterminant may also refer to a nucleotide construct and may becomprised in a vector. Alternatively, a genetic determinant may betransferred from one plant to another by chromosomal recombination aftercrossing said plants. A genetic determinant may in principle comprisegenetic material originating from one or more species.

In particular, genetic determinant as used herein refers to a singlegene or multiple genes, a QTL or a haplotype, that determines expressionof the seedless phenotype in a pepper plant.

A “gene” is defined herein as a hereditary unit consisting of a sequenceof DNA that occupies a specific location on a chromosome and thatcontains the genetic instruction for a particular characteristics ortrait in an organism.

A “locus” is defined herein as the position on a genetic map that agiven gene or any other genetic element or factor contributing to atrait occupies on a chromosome of a given species.

As used herein, the phrase “diploid individual” refers to an individualthat has two sets of chromosomes, typically one from each of its twoparents. However, it is understood that in some embodiments a diploidindividual can receive its “maternal” and “paternal” sets of chromosomesfrom the same single organism, such as when a plant is selfed to producea subsequent generation of plants.

The terms “chromosome” is meant to include, and thus used hereinsynonymously with, the terms “linkage group” and/or “chromosomeequivalent of linkage group”, respectively

As used herein, the term “heterozygous” means a genetic conditionexisting when different alleles reside at corresponding loci onhomologous chromosomes.

As used herein, the term “homozygous” means a genetic condition existingwhen identical alleles reside at corresponding loci on homologouschromosomes. Homozygosity is defined as absence of segregation afterselfing of an individual plant or, if crossed, absence of segregation inF1.

As used herein, the terms “hybrid”, “hybrid plant,” and “hybrid progeny”refers to an individual produced from genetically different or unlikeparents (e.g., a genetically heterozygous or mostly heterozygousindividual) (Rieger et al., 1968).

As used herein, the phrase “single cross F₁ hybrid” refers to an F₁hybrid produced from a cross between two inbred lines.

As used herein, the phrase “inbred line” refers to a geneticallyhomozygous or nearly homozygous population. An inbred line, for example,can be derived through several cycles of brother/sister breedings or ofselfing or in dihaploid production. In some embodiments, inbred linesbreed true for one or more phenotypic traits of interest. An “inbred”,“inbred individual”, or “inbred progeny” is an individual sampled froman inbred line.

As used herein, the term “dihaploid line”, refers to a stable inbredline issued from anther culture. Some pollen grains (haploid) cultivatedon specific medium and circumstances can develop plantlets containing nchromosomes. These plantlets are then “doubled” and contain 2nchromosomes. The progeny of these plantlets are named “dihaploid” andare essentially not segregating any more (stable).

As used herein, the term “progeny” refers to the descendant(s) of aparticular cross. Typically, progeny result from breeding of twoindividuals, although some species (particularly some plants andhermaphroditic animals) can be selfed (i.e., the same plant acts as thedonor of both male and female gametes). The descendant(s) can be, forexample, of the F₁, the F₂, or any subsequent generation.

As used herein, the terms “introgression”, “introgressed” and“introgressing” refer to the process whereby genes, a QTL or haplotypeof one species, variety or cultivar are moved into the genome of anotherspecies, variety or cultivar, by crossing those species. The crossingmay be natural or artificial. The process may optionally be completed bybackcrossing to the recurrent parent, in which case introgression refersto infiltration of the genes of one species into the gene pool ofanother through repeated backcrossing of an interspecific hybrid withone of its parents. An introgression may also be described as aheterologous genetic material stably integrated in the genome of arecipient plant.

“Genetic engineering”, “transformation” and “genetic modification” areall used herein as synonyms for the transfer of any kind of geneticinformation into the DNA of the target plant, usually but notexclusively the chromosomal DNA or genome, of another organism. Geneticengineering is one method of stably integrating heterologous geneticmaterial in the genome of a recipient plant and may include a processcomprises transforming cells or tissue of a plants with a DNArecombinant containing a heterologous DNA including a foreign nucleotidesequence encoding a gene or allelic variant thereof as well as theregulatory elements selected among those which are capable of causingthe stable integration of heterologous DNA in plant cells or tissue andof enabling the expression of foreign nucleotide sequences in plantcells or plant tissue.

As used herein, the phrase “genetic or molecular marker” refers to afeature of an individual's genome (e.g., a nucleotide or apolynucleotide sequence that is present in an individual's genome) thatis associated with one or more loci of interest. In some embodiments, agenetic marker is polymorphic in a population of interest, or the locusoccupied by the polymorphism, depending on context. Genetic markersinclude, for example, single nucleotide polymorphisms (SNPs), indels(i.e., insertions/deletions), simple sequence repeats (SSRs),restriction fragment length polymorphisms (RFLPs), random amplifiedpolymorphic DNAs (RAPDs), cleaved amplified polymorphic sequence (CAPS)markers, Diversity Arrays Technology (DArT) markers, and amplifiedfragment length polymorphisms (AFLPs), among many other examples.Genetic markers can, for example, be used to locate genetic locicontaining alleles on a chromosome that contribute to variability ofphenotypic traits. The phrase “genetic marker” can also refer to apolynucleotide sequence complementary to a genomic sequence, such as asequence of a nucleic acid used as probes.

A genetic or molecular marker can be physically located in a position ona chromosome that is within or outside of to the genetic locus withwhich it is associated (i.e., is intragenic or extragenic,respectively). Stated another way, whereas genetic markers are typicallyemployed when the location on a chromosome of the gene or of afunctional mutation, e.g. within a control element outside of a gene,that corresponds to the locus of interest has not been identified andthere is a very low rate of recombination between the genetic marker andthe locus of interest, the presently disclosed subject matter can alsoemploy genetic markers that are physically within the boundaries of agenetic locus (e.g., inside a genomic sequence that corresponds to agene such as, but not limited to a polymorphism within an intron or anexon of a gene). In some embodiments of the presently disclosed subjectmatter, the one or more genetic markers comprise between one and tenmarkers, and in some embodiments the one or more genetic markerscomprise more than ten genetic markers.

As used herein, the term “Restriction Fragment Length Polymorphism” or“RFLP” means a variation between individuals in DNA fragment sizes cutby specific restriction enzymes. Polymorphic sequences that result inRFLPs are used as markers on genetic linkage maps.

“Marker-based selection” is understood within the scope of the inventionto refer to e.g. the use of genetic markers to detect one or morenucleic acids from the plant, where the nucleic acid is associated witha desired trait to identify plants that carry genes, QTL or haplotypefor desirable (or undesirable) traits, so that those plants can be used(or avoided) in a selective breeding program.

“Microsatellite or SSRs (Simple sequence repeats) Marker” is understoodwithin the scope of the invention to refer to a type of genetic markerthat consists of numerous repeats of short sequences of DNA bases, whichare found at loci throughout the plant's genome and have a likelihood ofbeing highly polymorphic.

A “single nucleotide polymorphism” (SNP) is a DNA sequence variationoccurring when a single nucleotide, A, C, G, T, in the genome (or othershared sequences as mitochondrial DNA) differs between a set (paired)chromosomes of an individual or differs between members of a species.

“PCR (Polymerase chain reaction)” is understood within the scope of theinvention to refer to a method of producing relatively large amounts ofspecific regions of DNA or subset(s) of the genome, thereby makingpossible various analyses that are based on those regions.

“PCR primer” is understood within the scope of the invention to refer torelatively short fragments of single-stranded DNA used in the PCRamplification of specific regions of DNA.

“Polymorphism” is understood within the scope of the invention to referto the presence in a population of two or more different forms of agene, genetic marker, or inherited trait or a gene product obtainable,for example, through alternative splicing, DNA methylation, etc.

“Selective breeding” is understood within the scope of the invention torefer to a program of breeding that uses plants that possess or displaydesirable traits as parents.

“Tester” plant is understood within the scope of the invention to referto a plant of the genus Capsicum used to characterize genetically atrait in a plant to be tested. Typically, the plant to be tested iscrossed with a “tester” plant and the segregation ratio of the trait inthe progeny of the cross is scored.

“Probe” as used herein refers to a group of atoms or molecules which iscapable of recognising and binding to a specific target molecule orcellular structure and thus allowing detection of the target molecule orstructure. Particularly, “probe” refers to a labeled DNA or RNA sequencewhich can be used to detect the presence of and to quantify acomplementary sequence by molecular hybridization.

As used herein, the term “population” means a genetically homogeneous orheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term “variety” or “cultivar” means a group ofsimilar plants that by structural features and performance can beidentified from other varieties within the same species. The term“variety” as used herein has identical meaning to the correspondingdefinition in the International Convention for the Protection of NewVarieties of Plants (UPOV treaty), of Dec. 2, 1961, as Revised at Genevaon Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991. Thus,“variety” means a plant grouping within a single botanical taxon of thelowest known rank, which grouping, irrespective of whether theconditions for the grant of a breeder's right are fully met, can be i)defined by the expression of the characteristics resulting from a givengenotype or combination of genotypes, ii) distinguished from any otherplant grouping by the expression of at least one of the saidcharacteristics and iii) considered as a unit with regard to itssuitability for being propagated unchanged.

As used herein, the term “pepper” or “Capsicum” means any species,variety, cultivar, or population of the Capsicum genus.

A “cultivated Capsicum” plant is understood within the scope of theinvention to refer to a plant that is no longer in the natural state buthas been developed by human care and for human use and/or consumption.

As used herein, the term “breeding”, and grammatical variants thereof,refer to any process that generates a progeny individual. Breedings canbe sexual or asexual, or any combination thereof. Exemplary non-limitingtypes of breedings include crossings, selfings, doubled haploidderivative generation, and combinations thereof.

As used herein, the phrase “established breeding population” refers to acollection of potential breeding partners produced by and/or used asparents in a breeding program; e.g., a commercial breeding program. Themembers of the established breeding population are typicallywell-characterized genetically and/or phenotypically. For example,several phenotypic traits of interest might have been evaluated, e.g.,under different environmental conditions, at multiple locations, and/orat different times. Alternatively or in addition, one or more geneticloci associated with expression of the phenotypic traits might have beenidentified and one or more of the members of the breeding populationmight have been genotyped with respect to the one or more genetic locias well as with respect to one or more genetic markers that areassociated with the one or more genetic loci.

“Backcrossing” is understood within the scope of the invention to referto a process in which a hybrid progeny is repeatedly crossed back to oneof the parents. Different recurrent parents may be used in subsequentbackcrosses.

The following description is provided, alongside all chapters of thepresent invention, so as to enable any person skilled in the art to makeuse of said invention. Various modifications, however, will remainapparent to those skilled in the art, since the generic principles ofthe present invention have been defined specifically to provide a malesterile seedless pepper with unique characteristics as set forth below.

In the process of hybrid seed production male sterile inbreeds areusually used as the female parental line in order to increase theefficiency of hybrid production. Sterile pepper plants grow high andvery rapidly. At the top of the plant, they set very small, deformedseedless fruits. Spraying hormones on such plants has not succeeded ininducing “normal” fruit setting. But, as in many pepper traits there isa genetic variation to this phenomenon. Male sterile lines differ in theamount and quality of their seedless peppers.

Within the scope of the present invention pepper lines were discoveredthat were capable of setting qualitative, high yielding seedless fruits,particularly fruits that did not show deformations. Deformation in thiscontext means that a fruit shows an irregular and unusual form e.g., isnot uniform, not symmetric, or as irregular or shows no segmentations.

The size of an SLP fruit can be smaller than of a normal, seed-bearingfruit if compared on an otherwise identical genetic background. Theseed-bearing sibling can be up to 5-times larger than its SLP relative.However, it is possible by crossing into different genetic backgroundsto compensate against this smaller size and to obtain seedless pepperfruits which have the same or essentially the same size as a commercialsize sweet pepper fruit.

In particular, the SLP fruit at maturity may weigh between 2 grams and10 grams, particularly between 2.5 g and 5 g, or has a size of 1 cm to 4cm, particularly of 1.5 cm to 3 cm by 0.5 cm to 5 cm, particularly by 2cm to 4 cm in diameter.

The “seedless” trait is under the control of a genetic determinant andis permanently expressed in the pepper plants according to the presentinvention independent of artificial treatment with inducing agents suchas, for example, plant hormones or plant extracts, which are commonlyused in protected pepper cultivation in the greenhouse to artificiallyinduce parthenocarpy in pepper. The expression of the “seedless” traitin the plants according to the invention is also largely independent ofother exogenous factors including the pollination or fertilizationprocess, climatic conditions or seasonal variabilities.

As a result, the plants according to the present invention showexcellent fruit setting at all seasons that is also under unfavourablegrowing conditions such as those present in the winter season in theArava region in Israel (mediteranean climate), where the average mediumtemperature is in a range of between 4° C. and 14° C., particularlybetween 6° C. and 12° C.

In one embodiment of the invention, a male sterile seedless pepper (SLP)plant was developed which is characterized by its ability to setseedless fruit throughout the plant. The fruits are small in size suchthat they can typically be consumed in a single bite.

In particular, the isolated male sterile seedless pepper plant lineexhibits characteristic features including those selected from the groupconsisting of (i) excellent fruit setting at all seasons; (ii) strong &hardy plant; (iii) weak apical dominance as compared with non-sterilepeppers resulting in many side branches and ramifications of twigs; (iv)tendency to set two fruits per node in about 20% of nodes; (v) fairlyuniform size and shape; (vi) short intervals between nodes, approx. 10cm long; (vii), fruit production begins from the first nodes andprogresses along the length of the branches, similar to non-sterileplants; and, (viii) the flower has small anthers without pollen.

Further, the pepper fruits growing on said isolated plant line exhibitscharacteristic features including those selected from the groupconsisting of (i) seedless fruits; (ii) red pigmented in mature andgreen in premature (unripe) fruits; (iii) conic-like shape, i.e.,between bell and classic conic; (iv) size between about 2 to 4 cm longby about 3 to 4 cm in diameter; and (v) very sweet taste (Brix about 8to about 11°);

The present invention provides experimental evidence that suggests thatthe “seedless” trait shown by the plants of this invention aregenetically controlled. In particular, it appears that the “seedless”trait is multigenic and facultative. When these plants and varieties arepollinated, either by insect or by hand labor, fruit set and developmentoccur normally. However, under conditions in which pollination isdifficult or does not occur, these plants and varieties nevertheless setfruit that develops normally, whereas conventional plants and varietiesof the prior art might not set fruit at all, or the fruit will set butwill often be deformed and/or immediately begin to rot from the blossomend, and fruit development is poor at best, eventually being overtakenby the rot proceeding from the blossom end.

The “seedless” trait can be introgressed in any other plant or plantline of the Capsicum genus by a method selected from the groupconsisting of breeding, single trait conversion and transformation. Inparticular, the “seedless” trait can be introgressed into commercialpepper lines and varieties by methods known to those skilled in the artof plant breeding.

Commercial peppers are generally hybrids produced from the cross of twoparental lines (inbreds). The development of hybrids requires, ingeneral, the development of homozygous inbred lines, the crossing ofthese lines, and the evaluation of the crosses. Pedigree breeding andrecurrent selection breeding methods are used to develop inbred linesfrom breeding populations. Breeding programs combine the geneticbackgrounds from two or more inbred lines or various other germplasmsources into breeding pools from which new inbred lines are developed byselfing and selection of desired phenotypes. The new inbreds are crossedwith other inbred lines and the hybrids from these crosses are evaluatedto determine which of those have commercial potential. Plant breedingand hybrid development are expensive and labour and time-consumingprocesses.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding five or more generations ofselfing and selection is practiced: F1 to F2; F3 to F4; F4 to F5, etc. Asingle cross hybrid results from the cross of two inbred lines, each ofwhich has a genotype that complements the genotype of the other. Thehybrid progeny of the first generation is designated F1. In thedevelopment of commercial hybrids only the F1 hybrid plants are sought.Preferred F1 hybrids are more vigorous than their inbred parents. Thishybrid performance (hybrid vigor or heterosis), can be manifested inmany polygenic traits, including increased vegetative growth andincreased yield. Breeding in peppers can be accelerated by the use ofdouble haploids obtained by anther culture. Such a technique gives thepossibility to secure the process by producing pure lines in a shorterperiod of time than the regular pedigree breeding process. Plants withinthe Capsicum genus can be easily cross-pollinated. A trait is alsoreadily transferred from one pepper plant to another plant, includingpepper plants of different types using conventional breeding techniques,for example to further obtain commercial lines. The introgression of atrait into the elite line is for example achieved by recurrent selectionbreeding, for example by backcrossing. In this case, the elite line(recurrent parent) is first crossed to a donor inbred (the non-recurrentparent) that carries the trait, particularly the “seedless” traitaccording to the present invention. The progeny of this cross is thenmated back to the recurrent parent followed by selection in theresultant progeny for the trait. After three, preferably four, morepreferably five or more generations of backcrosses with the recurrentparent with selection for the trait, particularly the “seedless” traitaccording to the present invention, the progeny is heterozygous for thelocus harboring the resistance, but is like the recurrent parent formost or almost all other genes (see, for example, Poehlman & Sleper(1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles ofCultivar Development, Vol. 1: Theory and Technique, 360-376,incorporated herein by reference). Selection for the trait is carriedout after each cross. Male sterility is available in pepper. Inparticular genetic male sterility is widely used in commercial lines.e.g., sweet pepper lines (see for example Daskatoff S. (1972), while inchilli pepper also cytoplasmic male sterility (Peterson, 1958) is used.Male sterile pepper mutants and their utilization in heterosis breeding.Eucarpia, meetings on genetic and breeding. Turin 1971, 205-210).

Accordingly, in one embodiment, the present invention relates to amethod of producing a seedless pepper plant comprising the steps of

-   -   i) providing seeds of an inbred line of a F1 hybrid of a        seedless pepper plant which is male sterile as a female line and        a male-fertile (seed-bearing) pepper plant as a male line;    -   ii) germinating said seed and growing a mature, fertile plant        therefrom;    -   iii) inducing self-pollination of said plant grown under (ii),        growing fruits and harvesting the fertile seeds therefrom, and    -   iv) growing plants from the seeds harvested under iii) and,        optionally, selecting plants which grow seedless fruits.

Pepper is a self-pollinated species, so normally a plant can be easilyrecognized as seedless only if it is also sterile. Theoretically, these2 phenomena are not interconnected with each other in the sense of beinggenetically linked, but practically, parthenocarpy can be easilyidentified when the plant has the 2 traits together. Using fertileplants would require cutting open the fruit to determine whether seedsare present or not.

In one aspect of the invention, plants expressing the “seedless” traitand thus growing seedless pepper fruits, may be identified and selectedby simple visual observation of the flowers and identifying male sterileindividuals. After distinguishing the sterile plants, they arere-planted and estimated for their seedless fruit setting ability. Thiscan be achieved, for example by examining the plant for the followingphenotypic characteristics:

-   -   1. Fruit setting has started in the first nodes, as in fertile        plants.    -   2. Fruit setting occurs independently from the pollination        and/or fertilization process in all the seasons when also        fertile plants normally set.    -   3. Fruit setting occurs independently from the pollination        and/or fertilization process under unfavorable condition.

In the alternative, marker-assisted breeding may be employed to identifythose individuals where invention relevant loci and/or flanking markerloci or marker loci genetically linked thereto, have favorablegenotypes, particularly homozygous favorable genotypes.

Thus, markers can be developed by methods known to the skilled personand used to identify and select plants according to the presentinvention and as disclosed herein before with an allele or a set ofalleles of locus or loci representing the seedless trait.

There are several methods or approaches available, known to thoseskilled in the art, which can be used to identify and/or develop markersin linkage disequilibrium and/or linked to and/or located in the genomeregion where the gene(s), QTL or haplotype for the seedless traitresides, as well as markers that represent the actual causal mutationsunderlying the seedless trait. Without being fully exhaustive someapproaches, known by those skilled in the art, include:

-   -   candidate gene approach; candidate gene sequences or candidate        gene linked sequences can be searched for polymorphism        associated and/or genetically linked with the trait of interest        and once associated and/or genetically linked those polymorphism        can be used in marker assisted breeding applications.    -   Bulk segregant analysis (BSA) approach (Michelmore et al.,        1991); following phenotyping of a population, plants (usually        between 5-40) with contrasting phenotypes of the trait of        interest are grouped, with the groups forming bulks representing        the phenotypic extreme ends of the population. Then the bulks        are tested for the presence or absence of molecular marker        alleles. Since the bulks are supposed to contrast for alleles        contributing to the phenotypic extreme ends, any marker        polymorph between the bulks is a candidate genetically linked        marker, e.g. linked to the trait of interest and may be used in        marker assisted breeding applications, or, alternatively, may be        used to genetically map the trait of interest.    -   QTL mapping or association mapping approach: a population of        panel of plants may be characterized for the trait of interest        (phenotyped) and genotyped using markers preferably nicely        distributed over the entire genome. After obtained genotype data        and phenotype data there is searched for patterns of association        between the genotype data and phenotype data by joint analysis        of genotype and phenotype data using common QTL mapping and/or        association mapping software tools. Marker associated with the        trait of interest may subsequently be used in marker assisted        breeding applications.

Following QTL and/or association mapping, or following genetic mappingof makers linked to the trait of interest, other markers or genes knownto be located in the same genomic region (and thus genetically linked)may be identified and used to develop additional markers in the regionof interest and/or may be used in marker assisted breeding applications.

Genetically linked markers or marker sequences may also be used toisolate other nucleic acid sequences flanking those markers byhybridization, PCR, and/or ‘in-silico’ approaches. Those flankingnucleic acid sequences can be used to search for new and/or additionalpolymorphisms associated and/or genetically linked with the trait ofinterest which also can be used in marker assisted breeding populations.

Nucleic acid sequences associated and/or genetically linked to the traitof interest may also be used to in comparative genome and/or synthenymapping approaches to identify homologous region(s) and homologousand/or orthologous sequences and/or candidate genes.

In one embodiment, the present invention therefore relates to a geneticmarker that is genetically linked to the genetic determinant controllingthe “seedless” trait in a pepper plant according to the invention,particularly in a hybrid pepper plant designated Capsicum annuumAR07-F1-56-b; Capsicum annuum AR07-F1-87-b; Capsicum annuumAR07-F1-166-b; Capsicum annuum AR07-F1-171-X; and Capsicum annuumAR07-F1-172-X, grown from seeds deposited with NCIMB, Aberdeen AB21 9YA,Scotland, UK on May 26, 2008 under accession number NCIMB 41558, NCIMB41559, NCIMB 41560, NCIMB 41561 and NCIMB 41562, respectively, or a F2progeny thereof.

This genetic marker may be any marker selected from the group consistingof, but not limited to, restriction fragment length polymorphism (RFLP),random amplification of polymorphic DNA (RAPD), amplified restrictionfragment length polymorphism (AFLP), single sequence repeats (SSR) andsingle nucleotide polymorphisms SNPs or any nucleic acid sequenceassociated and/or genetically linked to the “seedless” trait.

The present invention also relates to a method of developing such amarker including those described herein above, particularly a markerthat is genetically linked to the genetic determinant controlling the“seedless” trait in a hybrid pepper plant designated Capsicum annuumAR07-F1-56-b; Capsicum annuum AR07-F1-87-b; Capsicum annuumAR07-F1-166-b; Capsicum annuum AR07-F1-171-X; and Capsicum annuumAR07-F1-172-X, grown from seeds deposited with NCIMB, Aberdeen AB21 9YA,Scotland, UK on May 26, 2008 under accession number NCIMB 41558, NCIMB41559, NCIMB 41560, NCIMB 41561 and NCIMB 41562, respectively, or a F2progeny thereof.

Also embraced by the present invention is the use of such a markeraccording to the invention for identifying other markers or genes knownto be located in the same genomic region (and thus genetically linked)and to develop additional markers in the region of interest and/or inmarker assisted breeding applications.

In a further embodiment, the present invention relates to a seedlesspepper plant comprising such a marker according to the invention whichis genetically linked to the genetic determinant controlling the“seedless” trait in said pepper plant, particularly a marker that isobtainable from a hybrid pepper plant designated Capsicum annuumAR07-F1-56-b; Capsicum annuum AR07-F1-87-b; Capsicum annuumAR07-F1-166-b; Capsicum annuum AR07-F1-171-X; and Capsicum annuumAR07-F1-172-X, grown from seeds deposited with NCIMB, Aberdeen AB21 9YA,Scotland, UK on May 26, 2008 under accession number NCIMB 41558, NCIMB41559, NCIMB 41560, NCIMB 41561 and NCIMB 41562, respectively, or from aF2 progeny thereof, particularly by applying a method as disclosedherein.

Marker-based selection may already be used in the early phases of inbreddevelopment, often in combination with screening methods which are basedlargely on phenotypic characteristics that can be determined visuallyand are related to the trait of interest, i.e. the “seedless” trait, andfurther to key performance indices such as, for example, plant vigor,length of internodes, ramifications, insect resistance, virusresistances such as TMV (Tobacco Mosaic Virus) and TSWV (Tomato SpottedWilt Virus), etc., which are relevant for the suitability of the plantto be utilized in commercial hybrid production. Selection may also bebased on molecular markers, which may or may not be linked to traits ofinterest.

In particular, marker-based selection may be applied in combination withor followed by a phenotypic selection to identify those individualswhere all of the invention relevant loci have heterozygous or homozygousfavorable genotypes.

There are several types of molecular markers that may be used inmarker-based selection including, but not limited to, restrictionfragment length polymorphism (RFLP), random amplification of polymorphicDNA (RAPD), amplified restriction fragment length polymorphism (AFLP),single sequence repeats (SSR) and single nucleotide polymorphisms SNPs.

RFLP involves the use of restriction enzymes to cut chromosomal DNA atspecific short restriction sites, polymorphisms result from duplicationsor deletions between the sites or mutations at the restriction sites.

RAPD utilizes low stringency polymerase chain reaction (PCR)amplification with single primers of arbitrary sequence to generatestrain-specific arrays of anonymous DNA fragments. The method requiresonly tiny DNA samples and analyses a large number of polymorphic loci.

AFLP requires digestion of cellular DNA with a restriction enzyme(s)before using PCR and selective nucleotides in the primers to amplifyspecific fragments. With this method, using electrophoresis techniquesto visualize the obtained fragments, up to 100 polymorphic loci can bemeasured per primer combination and only small DNA sample are requiredfor each test.

SSR analysis is based on DNA micro-satellites (short-repeat) sequencesthat are widely dispersed throughout the genome of eukaryotes, which areselectively amplified to detect variations in simple sequence repeats.Only tiny DNA samples are required for an SSR analysis.

SNPs use PCR extension assays that efficiently pick up point mutations.The procedure requires little DNA per sample. One or a combination ofthe above methods may be used in a typical marker-based selectionbreeding program.

Currently, the most preferred method of achieving amplification ofnucleotide fragments that span a polymorphic region of the plant genomeemploys the polymerase chain reaction (“PCR”) (Mullis et al., ColdSpring Harbor Symp. Quant. Biol. 51:263 273 (1986)), using primer pairsinvolving a forward primer and a backward primer that are capable ofhybridizing to the proximal sequences that define a polymorphism in itsdouble-stranded form.

Basically, the method of PCR amplification involves use of a primer or apair of primers comprising two short oligonucleotide primer sequencesflanking the DNA segment to be amplified or adapter sequences ligated tosaid DNA segment. Repeated cycles of heating and denaturation of the DNAare followed by annealing of the primers to their complementarysequences at low temperatures, and extension of the annealed primerswith DNA polymerase. The primers hybridize to opposite strands of theDNA target sequences. Hybridization refers to annealing of complementaryDNA strands, where complementary refers to the sequence of thenucleotides such that the nucleotides of one strand can bond with thenucleotides on the opposite strand to form double stranded structures.The primers are oriented so that DNA synthesis by the polymeraseproceeds bidirectionally across the nucleotide sequence between theprimers. This procedure effectively doubles the amount of that DNAsegment in one cycle. Because the PCR products are complementary to, andcapable of binding to, the primers, each successive cycle doubles theamount of DNA synthesized in the previous cycle. The result of thisprocedure is exponential accumulation of a specific target fragment,that is approximately 2<n>, where n is the number of cycles.

Through PCR amplification millions of copies of the DNA segment flankedby the primers are made. Differences in the number of repeated sequencesor insertions or deletions, which are located between the flankingprimers in different alleles are reflected in length variations of theamplified DNA fragments. These variations can be detected, for example,by electrophoretically separating the amplified DNA fragments on gels orby using capillary sequencer. By analyzing the gel or profile, it can bedetermined whether the plant contains the desired allele in a homozygousor heterozygous state or whether the desired or undesired allele isabsent from the plant genome.

Sequence variations among different alleles which do not result inlength variation, like SNPs can be determined (genotyped) by a diverserange of genotyping methods which could be hybridization-based,enzyme-based, or by other post-amplification methods based on physicalproperties of the DNA, as well as by sequencing.

Alternative methods may be employed to amplify fragments, such as the“Ligase Chain Reaction” (“LCR”) (Barany, Proc. Natl. Acad. Sci. (U.S.A.)88:189 193 (1991)), which uses two pairs of oligonucleotide probes toexponentially amplify a specific target. The sequences of each pair ofoligonucleotides are selected to permit the pair to hybridize toabutting sequences of the same strand of the target. Such hybridizationforms a substrate for a template-dependent ligase. As with PCR, theresulting products thus serve as a template in subsequent cycles and anexponential amplification of the desired sequence is obtained.

LCR can be performed with oligonucleotides having the proximal anddistal sequences of the same strand of a polymorphic site. In oneembodiment, either oligonucleotide will be designed to include theactual polymorphic site of the polymorphism. In such an embodiment, thereaction conditions are selected such that the oligonucleotides can beligated together only if the target molecule either contains or lacksthe specific nucleotide that is complementary to the polymorphic sitepresent on the oligonucleotide. Alternatively, the oligonucleotides maybe selected such that they do not include the polymorphic site (see,Segev, PCT Application WO 90/01069).

A further method that may alternatively be employed is the“Oligonucleotide Ligation Assay” (“OLA”) (Landegren et al., Science241:1077 1080 (1988)). The OLA protocol uses two oligonucleotides thatare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. OLA, like LCR, is particularly suited for thedetection of point mutations. Unlike LCR, however, OLA results in“linear” rather than exponential amplification of the target sequence.

Still another method that may alternatively be employed is the “InvaderAssay” that uses a structure-specific flap endonuclease (FEN) to cleavea three-dimensional complex formed by hybridization of allele-specificoverlapping oligonucleotides to target DNA containing a singlenucleotide polymorphism (SNP) site. Annealing of the oligonucleotidecomplementary to the SNP allele in the target molecule triggers thecleavage of the oligonucleotide by cleavase, a thermostable FEN.Cleavage can be detected by several different approaches. Most commonly,the cleavage product triggers a secondary cleavage reaction on afluorescence resonance energy transfer (FRET) cassette to release afluorescent signal. Alternatively, the cleavage can be detected directlyby use of fluorescence polarization (FP) probes, or by massspectrometry. The invasive cleavage reaction is highly specific, has alow failure rate, and can detect zeptomol quantities of target DNA.While the assay traditionally has been used to interrogate one SNP inone sample per reaction, novel chip- or bead-based approaches have beentested to make this efficient and accurate assay adaptable tomultiplexing and high-throughput SNP genotyping.

Nickerson et al. have described a nucleic acid detection assay thatcombines attributes of PCR and OLA (Nickerson et al., Proc. Natl. Acad.Sci. (U.S.A.) 87:8923 8927 (1990)). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

Schemes based on ligation of two (or more) oligonucleotides in thepresence of a nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, arealso known (Wu et al., Genomics 4:560 569 (1989)), and may be readilyadapted to the purposes of the present invention.

A molecular marker may thus be DNA fragment amplified by PCR, e.g. a SSRmarker or a RAPD marker. The presence or absence of an amplified DNAfragment may be indicative of the presence or absence of the traititself or of a particular allele of the trait. When using SSR markers, adifference in the length of an amplified DNA fragment may be indicativeof the presence of a particular allele of a trait, and thus enables todistinguish between different alleles of a trait.

In a specific embodiment of the invention simple sequence repeat (SSR)markers are used to identify invention-relevant alleles in the parentplants and/or the ancestors thereof, as well as in the progeny plantsresulting from a cross of said parent plants. Simple sequence repeatsare short, repeated DNA sequences and present in the genomes of alleukaryotes and consists of several to over a hundred repeats of a givennucleotide motif. Since the number of repeats present at a particularlocation in the genome often differs among plants, SSRs can be analyzedto determine the absence or presence of specific alleles.

In another embodiment of the invention SNP markers are used to identifyinvention-relevant alleles in the parent plants and/or the ancestorsthereof, as well as in the progeny plants resulting from a cross of saidparent plants.

Marker analysis can be done early in plant development using DNA samplesextracted from leaf tissue of very young plants or from seed. Thisallows to identify plants with a desirable genetic make-up early in thebreeding cycle and to discard plants that do not contain the desired,invention-relevant alleles prior to pollination thus reducing the sizeof the breeding population and reducing the requirements of phenotyping.

Further, by using molecular markers, a distinction can be made betweenhomozygous plants that carry two copies of the desired,invention-relevant allele at invention-relevant loci and heterozygousplants that carry only one copy and plants that do not contain any copyof the favourable allele(s).

Molecular markers may be used in marker-assisted-selection and/or anyother methods wherein plants having or have not the “seedless” trait aretraced. The markers may be either trans, or cis markers. A trans markerindicates a polymorphism resulting from introgression of exogenous(donor) DNA into a recipient plant's genome, which polymorphism islinked in cis with the recipient genome, i.e. linked with the oppositeallele. Thus, cis markers are linked with the allele of interest, whiletrans markers are linked with the opposite allele (from the recipient).

To determine the utility of the inbred line and its potential togenetically contribute to the hybrid progeny a test-cross is made withanother inbred line, and the resulting progeny phenotypically evaluated.Traits that may be recorded commonly involve traits that are related tofruit shape and fruit characteristics such as pointed or non pointedfruit, pungent or non pungent, red, yellow or orange. Plantcharacteristics as length of internodes, growing power and ramificationsare also considered together with specific virus resistances such as TMV(Tobacco Mosaic virus) and TSWV (Tomato Spotted wilt virus).

For genotyping, or association mapping DNA is extracted from suitableplant material such as, for example, leaf tissue. In particular, bulksof leaves of a plurality of plants are collected. DNA samples aregenotyped using a plurality of polymorphic SSR's, SNPs or any othersuitable marker-type covering the entire pepper genome.

Joint-analysis of genotypic and phenotypic data can be performed usingstandard software.

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlyclaimed subject matter.

Example 1 Isolation of a Seedless Pepper Line

1.1 Growth and Cultivation Conditions: The SLPs of the present inventionwere grown at Netiv HaHasara and Zofar (Israel) for two growing seasons,i.e., spring and summer 2006; and fall and winter 2006-2007. As in mostplants, especially peppers, the described characteristics are related togrowth, seasons and conditions. It can be assumed that under otherconditions, the plant characteristics may well differ. The followingexample is presented:

Netiv HaHasara conditions: unheated plastic covered greenhouse, coveredby shading net (40%, from May); sandy soil; sowing in February 2006;2,500 plants per 1,000 m²; Spanish-style side support cane; non-pruningof fruits and plants, as in accepted commercial practice.

Zofar conditions: unheated net house while using double nets, one whitenetting (25 MESH, 20% shading) throughout season and black netting, 40%shading for the first 40 days, and the last two months of the growingseason (i.e., February and March), sandy soil; sowing in July 2006;3,500 plants per 1,000 m²; Spanish-style side support cane; non-pruningof fruits and plants, as in accepted commercial practice.

1.2 Pedigree Information: The pedigree of one such seedless plant is asshown in the scheme below:

The source of P1 and P2 are F2 seeds of commercial varieties grown infield L3. The population of P2 (the plot L3-188) was divided bysterility. P1 and P2 were selected according to their strong plants andquite good quality. P1 was made from seeds from a fertile plant, No 4,in the plot “L3-161” and P2 is a bulk of seeds from many fertile plantsin the plot L3-188. Both were red pepper type. The hybrid “B5-F1-217-b”was made by these 2 plants, where P1 was the male parent and P2 was thefemale. The hybrid was grown (15 plants) in the field PC in plot 122,and F2 seeds were collected from all the plants. These F2 seeds weresowed in the field L5 in plot 155. This population was split bysterility. Among many other sterile plants plant number 1 wasdistinguished by its ability to set seedless small fruit all along theplant. The plant was vegetatively multiplied and the offspring weregrown in the fall-winter season. In this season the performance of theseplants was very similar to spring-summer.

1.3 Plant and Fruit Characteristics: The experiments provided fruits andplants (See pictures 1 and 2), defined by the following: Seedless fruitcharacterized by (i) seedless; (ii) Red pigmented in mature and darkgreen pigmented in premature (unripe) fruits; (iii) conic-like shape,i.e., between bell and classic conic; (iv) size is between about 2 to 4cm long by about 3 to 4 cm in diameter; and (v) very sweet taste (Brixabout 8 to about 11°). The seedless plant is characterized by featuresselected from a group comprising inter alia of (i) Excellent fruitsettings at all seasons; (ii) Strong & hardy plant; (iii) Many sidebranches, ramifications of twigs, weak apical dominance; (iii) Tendencyto set two fruit per node in about 20% of nodes; (iv) Fairly uniformsize and shape; (v) Short intervals between nodes, approx. 10 cm long;(vi), fruit production begins from the first nodes and progresses alongthe length of the branches, similar to non-sterile plants; and, (vii)the flower has small anthers without pollen.

Example 2 Genetics of the “Seedless” Trait

To demonstrate that the “seedless” trait, discovered in the “one-bite”pepper type, is an independent trait controlled by a genetic determinantand can therefore be passed on genetically to many different peppertypes, it is shown that the trait will be present in offspring ofpopulations made by crossing the seedless plant with other pepper typeplants.

2.1 Breeding History: In spring 2007, the SLP variety 255-1 was crossedwith 8 varieties of different types; Blocky, Kapya and Conic. Thehybrids were grown in autumn/winter 2007-2008 and F2 seeds wereharvested from 10 F1 plants.

The Hybrid Name Description Male Name Female Name 1 AR07-F1-56-b HybridNJ06-F3-385-4 AR06-F3-255-1 255-1 × Kapya (Kapya) 2 AR07-F1-87-b HybridNA06- F3-14-4 AR06-F3-255-1 255-1 × Kapya (Kapya) 3 AR07-F1-166-b HybridNJ06-F3-378-1 AR06-F3-255-1 255-1 × Conic (conic) 4 AR07-F1-171-x HybridAV05-F1-7158-TT AR06-F3-255-1 255-1 × Blocky (Blocky) 5 AR07-F1-172-xHybrid AV05-F1-7181-TT AR06-F3-255-1 255-1 × Blocky (Blocky)

Example 3 Recovery of the “Seedless” Trait from the Hybrids

3.1 Experimental Design: The 5 deposited hybrids were grown from samplesof the deposited seed, 10 individual plants for each (50 plants intotal). F2 seeds from the individual plants were obtained by selfpollination. The F2 seeds were harvested from the plants and keptseparate. 55 F2 seeds from each F1 plant were sown (2,750 seeds intotal, 550 seeds per hybrid family). The target was to achieve 100−130sterile F2 plants from each deposited hybrid family (˜25% of 550). Thetrial was designed so that each F2 offspring was separatelytransplanted. Additionally, as a control, F2 seeds from the originalhybrid, where the SLP trait was discovered in the offspring, were alsosown: 600 seeds from 3 F1 plants, 200 seeds from each. The F2 Plants arethan analyzed for expression of a male sterile (MS) trait and theseedless pepper (SLP) trait. Both traits segregate separately, howeverthe MS trait is necessary to have the SLP trait phenotypicallyexpressed. While in the female line (SLP line) the MS trait ishomogenous recessive, in the F1 hybrid it is heterogenic (the male linesare all homogenous male fertile).

As a backup, additional seeds were sown a week later. This sowing cycleincluded the families sown in the first cycle (in case the germinationis insufficient) and F2 families from other hybrids to enrich thepotential variation.

About 1 to 2 month later, the seedlings are transplanted to pots andcontinue to grow in the nursery. The screening and selection of seedlesssterile plants is done by simple observation of the flowers. Afterdistinguishing the sterile plants, they are planted in net house (whitenet 50 mash to avoid cross pollination by insects) at a density of 2,500plants/1,000 m². Parthenocarpic phenotyping is done 2 weeks later.

3.2 Results: The male sterile trait (MS), which is comprised in the F2seeds, is expressed with an average rate of 27% (not far from thetheoretical rate 25% which is expected for a monogenic recessive trait).The SLP trait can only be phenotypically expressed in a MS background.The average rate of plants expressing the SLP trait in a population ofMS plants is 23%. Although the SLP trait is not dependent onenvironmental conditions, the degree of expression can be influenced bythe genetic background of the male line. It ranges from 6% (originalhybrid) up to 36% in the hybrid AR07-F1-56-b made by crossing the SLPplant (AR06-F3-255-1) with Kapya line. In all trials based on thedeposited lines the SLP trait could be consistently recovered. The dataindicate that the SLP trait is a complex trait with at least 2 primarygenetic components involved.

In all SLP plants, 100% of the fruits were seedless, and of all thesefruits were 100% seedless (no ovules were fertilized to become seedbearing). In negative fertile control only very occasionally someindividual fruits on a plant were discovered to be seedless; howeverthis is the result of climate effects or irregular pollination but not agenetic phenomenon.

Deposits

The following seed samples of Capsicum annuum lines were deposited withNCIMB, Aberdeen AB21 9YA, Scotland, UK on May 26, 2008 under theprovisions of the Budapest Treaty:

Capsicum annuum seed line designation Deposition date Accession NoAR07-F1-56-b 26 May 2008 NCIMB 41558 AR07-F1-87-b 26 May 2008 NCIMB41559 AR07-F1-166-b 26 May 2008 NCIMB 41560 AR07-F1-171-X 26 May 2008NCIMB 41561 AR07-F1-172-X 26 May 2008 NCIMB 41562

LIST OF REFERENCES

-   Sjut V, Bangerth F (1982) “Induced parthenocarpy: a way of changing    the levels of endogenous hormones in tomato fruits (Lycopersicon    esculentum Mill.): 1. Extractable hormones.” Plant Growth Regul 1:    243-251-   Kim I S, Okubo H, Fujieda K. 1992. “Endogenous levels of IAA in    relation to parthenocarpy in cucumber (Cucumis sativus L.).”    Scientia Horticulturae 52: 1-8.-   Beyer E M, Quebedeaux B. 1974. “Parthenocarpy in cucumber: mechanism    of action of auxin transport inhibitors.” Journal of the American    Society for Horticultural Science 99: 385-390.-   Heuvelink and Körner “Parthenocarpic Fruit Growth Reduces Yield    Fluctuation and Blossom-end Rot in Sweet Pepper”, Annals of Botany.    88(1):69-74, (2001)-   Rieger, R. et al, A “Glossary of Genetics and Cytogenetics,    Classical and Molecular Book Microbes Human”, George Allen & Unwin    Ltd.; Springer-Verlag, London; Berlin, UK; Germany: 1968. 3rd ed.    (rev.), 507 pp-   International Convention for the Protection of New Varieties of    Plants (UPOV treaty), of Dec. 2, 1961, as Revised at Geneva on Nov.    10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991-   Poehlman & Sleper (1995) “Breeding Field Crops”, 4th Ed., 172-175;    Fehr (1987) Principles of Cultivar Development, Vol. 1: Theory and    Technique, 360-376,-   Daskaloff S. 1972. Male sterile pepper (C. annuum L.) mutants and    their utilization in heterosis breeding. Eucarpia, Meeting on    Genetics and Breeding of Capsicum. (Torino, Italy), pp. 205-210.-   Peterson, 1958 “Cytoplasmically inherited male-sterility in    Capsicum.” Am. Nat. 92:111-119.-   Michelmore, R. W., Para, I., and Kasseri, R. V. (1991).    “Identification of markers linked to disease-resistance genes by    bulked segregant analysis: a rapid method to detect markers in    specific genomic regions by using segregating populations.” Proc.    Natl. Acad. Sci., USA, 88: 9828-9832-   Mullis et al., “Specific enzymatic amplification of DNA in-vitro the    polymerase chain reaction.” Cold Spring Harbor Symp. Quant. Biol.    51:263-274 (1986)-   Barany, “Genetic disease detection and DNA amplification using    cloned thermostable ligase” Proc. Natl. Acad. Sci. (U.S.A.) 88:189    193 (1991)-   Landegren et al., “A ligase-mediated gene detection technique”    Science 241:1077 1080 (1988)-   Nickerson et al., “Automated DNA diagnostics using an ELISA-based    oligonucleotide ligation assay” Proc. Natl. Acad. Sci. (U.S.A.)    87:8923-8927 (1990)-   Wu et al., “The ligation amplification reaction (LAR)—amplification    of specific DNA sequences using sequential rounds of    template-dependent ligation.” Genomics 4:560-569 (1989)-   PCT Application WO 90/01069

The invention claimed is:
 1. A male sterile pepper plant comprising aseedless trait, which grows seedless fruits independent of thepollination and fertilization process, wherein the pepper plant is aCapsicum annuum plant, and wherein the seedless trait is controlled by agenetic determinant and is independent of exogenous treatment withparthenocarpy-inducing plant hormones, and wherein the plant comprisesthe genetic determinant conferring the seedless trait of the hybridpepper designated AR07-F1-166-b, representatitve seeds of said hybriddeposited with the NCIMB, Aberdeen AB21 9YA, Scotland, UK on May 26,2008 under accession number
 41560. 2. The pepper plant according toclaim 1, wherein said seedless fruit is characterized by being at least95% seedless.
 3. The pepper plant according to claim 2, wherein saidseedless fruit is characterized by being 100% seedless.
 4. The pepperplant according to claim 2, wherein at least 60% of the fruits grown onsaid plant are at least 95% seedless.
 5. The pepper plant according toclaim 1, wherein at least 40% of the fruits grown on said plant are atleast 100% seedless.
 6. A pepper plant according to claim 1, whereinfruit setting starts at the first nodes of a branch and progresses alongthe length of the entire branch.
 7. A pepper plant according to claim 6,wherein said plant grows two fruits per node in about 20% of the nodes.8. The pepper plant according to claim 1, which grows seedless fruitswhich are edible and suitable for a use selected from the groupconsisting of as fresh produce, as fresh cut produce, for processing andfor the production of preserved food.
 9. The plant according to claim 8,wherein said plant grows a sweet pepper selected from the groupconsisting of a dolce-type pepper, a bell pepper, a big rectangularpepper, a conical pepper, a long conical pepper and a blocky-typepepper.
 10. The plant according to claim 9, wherein a mature fruit ofsaid plant is evergreen, yellow, orange, ivory, brown, purple or red.11. The plant according to claim 10, which grows pepper fruits which arered pigmented in the mature and dark green pigmented in the premature orunripe stage.
 12. The plant according to claim 1, wherein said plant isan inbred, a dihaploid or a hybrid.
 13. Plant material obtained from aplant according to claim 1, wherein said plant material comprises thegenetic determinant conferring the seedless trait.
 14. Plant parts of aplant according to claim 1, wherein said plant parts comprise thegenetic determinant conferring the seedless trait.
 15. A fruit of aplant according to claim
 1. 16. A fruit according to claim 15 which is aprocessed fruit.